A pougt75c1 gene and application thereof in regulating content of plant fatty acid and stilbene compound

Abstract

This invention discloses a PoUGT75C1 gene and its application in regulating the content of fatty acids and stilbene compounds in plants, belonging to the field of genetic engineering technology. The nucleotide sequence of the CDS region of the PoUGT75C1 gene is shown in SEQ ID NO.2. This invention verifies the regulatory effect of the PoUGT75C1 gene on stilbene compounds and fatty acids in plants using gene silencing and overexpression techniques. Specifically, silencing the PoUGT75C1 gene increases the accumulation of stilbene compounds, while both overexpression and silencing of the PoUGT75C1 gene increase fatty acid content and promote seed fatty acid accumulation. This invention provides a theoretical basis and genetic resources for the high-value utilization of peony germplasm resources and also offers new ideas for improving the quality of woody oil crops.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a PoUGT75C1 gene and its application in regulating the content of fatty acids and succinate compounds in plants. Background Technology

[0002] Peony fruit is an aggregate follicle, and the mature seeds are black, mostly round or oval in shape. As a novel woody oilseed resource, its seeds are rich in oil (>20%), of which unsaturated fatty acids account for more than 90%, polyunsaturated fatty acids (PUFAs) account for more than 70%, and α-linolenic acid (ALA) accounts for about 40%. In 2011, peony seed oil was approved as a new resource food by the former Ministry of Health of China, marking a major transformation of this traditional ornamental and medicinal plant resource into a functional edible oil.

[0003] In addition to its rich oil content, peony seeds are also rich in diverse secondary metabolites, mainly including monoterpenoid glycosides, arbutin, flavonoids, and triterpenoids, exhibiting a significant tissue-specific distribution: the total phenol content in the seed coat is as high as 139.49 mg / g, while that in the kernel is only 3.04 mg / g. Arbutin compounds are specifically enriched in the seed coat, accounting for more than 16.7% of the total content, and the total content of arbutin in the seed coat extract is as high as 75%. Flavonoids and monoterpenoid glycosides are more abundant in the kernel. This tissue-specific distribution pattern lays the material foundation for the intensive processing and utilization of peony seeds. The polyphenols, monoterpenoid glycosides, and other secondary metabolites abundant in peony seeds endow them with diverse biological activities. Existing studies have shown that its extracts have various pharmacological activities such as antioxidant, hypoglycemic, anti-radiation, and anti-tumor effects, and the seed coat is significantly superior to the kernel in all activity evaluations, demonstrating higher development and utilization value.

[0004] Plant glycosyltransferases (UGTs) possess diverse biological functions, including participation in plant hormone balance regulation, detoxification responses, defense responses, and the synthesis of secondary metabolites. With the deepening research on UDP-glucose (flavonoid glucosyltransferase) genes (UFGTs), their role in flavonoid glycosylation modification has been validated in various plants. In food crops, rice GSA1 catalyzes the synthesis of flavonoid glycosides and lignin through glycoside transfer. Under abiotic stress, it enhances stress resistance by reducing lignin synthesis and promoting flavonoid accumulation, while simultaneously regulating grain size. OsUGT706C2 specifically catalyzes the 7-O-glycosylation of apigenin, luteolin, and kaempferol. In the model plant Arabidopsis thaliana, AtUGT79B2 and AtUGT79B3 are responsible for rhamnylation at the 3-O position of anthocyanins, while AtUGT84A2 is involved in the transfer of 1-O-sinosinolate glucose to anthocyanins; mutations in AtUGT84A2 lead to a reduction of approximately 75% in the content of sinosylated anthocyanins. Yin et al. identified six UDP-glycosyltransferase genes (UGT72X4, UGT72Z3, UGT73C20, UGT88A13, UGT88E19, and UGT92G4) with flavonol glycosylation activity in soybean. These enzymes are active against flavonols, isoflavones, flavones, and flavanol aglycones, exhibiting different kinetic properties. Among them, UGT72X4, UGT72Z3, and UGT92G4 are flavonol-specific UGTs, while UGT73C20 and UGT88E19 are active against both flavonols and isoflavone aglycones. In particular, UGT88A13 is active against epicatechin, but inactive against flavonol aglycone kaempferol and quercetin.

[0005] Recent studies have also revealed novel functions of UGTs in metabolic cross-regulation. The alfalfa MtUGT84A1 mutant can glycosylate quercetin, apigenin, and luteolin, utilizing UDP-glucose and UDP-glucuronic acid as sugar donors; its mutants exhibit stunted growth and pigment deficiencies. In tea plants, the expression of CsUGT84J2 and CsUGT74Y1 can disrupt auxin and flavonol homeostasis, promoting plant growth. These studies indicate that UGTs not only participate in secondary metabolism but may also influence primary metabolism by regulating hormone homeostasis.

[0006] In peonies, PhUGT78A22 has been shown to catalyze secondary glycosylation of anthocyanins during petal spot formation. Wang et al. further revealed that the formation of bright red flower color in Japanese peony varieties is determined by pelargonidin-3-O-glucoside, and identified two key glycosyltransferase genes, PsUGT78A27 and PsUGT75L45, responsible for glycosylation modification of bright red and pink flower colors, respectively. However, research on UGT genes in peony seeds remains scarce. Summary of the Invention

[0007] The purpose of this invention is to provide a PoUGT75C1 gene and its application in regulating the content of fatty acids and succinate compounds in plants, so as to solve the problems existing in the prior art.

[0008] To achieve the above objectives, the present invention provides the following solution: This invention provides a PoUGT75C1 gene, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0009] The present invention also provides a protein encoded by the above-mentioned PoUGT75C1 gene, the amino acid sequence of which is shown in SEQ ID NO.3.

[0010] The present invention also provides a recombinant vector, wherein the recombinant vector is a recombinant vector that overexpresses the above-mentioned PoUGT75C1 gene, or a recombinant vector that silences the above-mentioned PoUGT75C1 gene.

[0011] The present invention also provides a recombinant microorganism containing the above-mentioned recombinant vector.

[0012] This invention also provides the use of the above-mentioned PoUGT75C1 gene, the above-mentioned protein, the above-mentioned recombinant vector, or the above-mentioned recombinant microorganism in any of the following: (1) Regulate the fatty acid content in plant seeds; (2) Preparation of formulations to regulate the fatty acid content in plant seeds; (3) Cultivate plants with high fatty acid content; (4) Regulate the content of erucic acid compounds in plant seed coats; (5) Preparation of formulations to regulate the content of zirconia compounds in plant seed coats; (6) Cultivate plants with high content of succinate compounds.

[0013] Furthermore, the method for regulating the fatty acid content in plant seeds includes the step of transferring the PoUGT75C1 gene into plants to stably overexpress it, thereby increasing the fatty acid content of plant seeds; or, the step of silencing the PoUGT75C1 gene in plants to increase the fatty acid content of plant seeds. Alternatively, the method for regulating the content of erucic acid compounds in plant seed coats may include the step of silencing the PoUGT75C1 gene in plants to increase the content of erucic acid compounds in plant seed coats.

[0014] Further, the fatty acids include C16:0, C18:0, C18:1, C18:2, C18:3, C20:1, C20:2, and C22:1; the ursine compounds include resveratrol and Suffruticosol B.

[0015] Optionally, the plants include peony and Arabidopsis thaliana.

[0016] The present invention also provides a method for increasing the fatty acid content in plant seeds, comprising the step of transferring the PoUGT75C1 gene into a plant and stably overexpressing it to increase the fatty acid content of plant seeds; or, the step of silencing the PoUGT75C1 gene in a plant to increase the fatty acid content of plant seeds. The nucleotide sequence of the CDS region of the PoUGT75C1 gene is shown in SEQ ID NO.2.

[0017] The present invention also provides a method for increasing the content of erucic acid compounds in plant seed coats, including the step of silencing the PoUGT75C1 gene in plants to increase the content of erucic acid compounds in plant seed coats; The nucleotide sequence of the CDS region of the PoUGT75C1 gene is shown in SEQ ID NO.2.

[0018] The present invention discloses the following technical effects: This invention provides a PoUGT75C1 gene from peony, the nucleotide sequence of which is shown in SEQ ID NO.1. This invention utilizes gene silencing and overexpression techniques to verify the regulatory effects of the PoUGT75C1 gene on stilbene compounds and fatty acids in plants. Specifically, silencing the PoUGT75C1 gene increases the accumulation of stilbene compounds (RE and SB), and silencing the PoUGT75C1 gene increases the content of fatty acids (C16:0, C18:0, C18:1, C18:2, ALA). Overexpression of the PoUGT75C1 gene increases the content of fatty acids (C16:0, C18:0, C18:1, C18:2, C18:3, C20:1, C20:2, C22:1), thereby promoting the accumulation of fatty acids in seeds. This invention provides a theoretical basis and gene resources for the high-value utilization of peony germplasm resources and also offers new ideas for the quality improvement of woody oil crops. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 SDS-PAGE analysis results of PoUGT75C1 protein expression under different induction conditions; Figure 2 The results are for the purified PoUGT75C1 protein; A represents the SDS-PAGE result, and B represents the Western Blot result. Figure 3 Results of in vitro enzyme activity analysis of PoUGT75C1 protein; Figure 4 Subcellular localization results for PoUGT75C1 protein; scale bar = 5 μm; Figure 5 RT-qPCR results validated the expression level of the PoUGT75C1 gene in the seeds of gene-silenced plants. Figure 6 The results show the determination of stilbene compounds in the seed coat of gene-silenced plants; among them, resveratrol: RE; Suffruticosol B: SB; Figure 7 The results show the fatty acid content in the seeds of gene-silenced plants; palmitic acid: PA; stearic acid: SA; oleic acid: OA; linoleic acid: LA; α-linolenic acid: ALA. Figure 8 RT-qPCR results validated the expression level of the PoUGT75C1 gene in Arabidopsis thaliana overexpression lines. Figure 9 The results show the fatty acid content in the seeds of Arabidopsis thaliana overexpression lines; palmitic acid: C16:0; stearic acid: C18:0; oleic acid: C18:1; linoleic acid: C18:2; linolenic acid: C18:3; eicosapentaenoic acid: C20:1; eicosadienoic acid: C20:2; erucic acid: C22:1. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0027] The inventors initially used seeds from different developmental stages as materials to perform metabolomics analysis, and based on transcriptomics and broad-target metabolomics, they identified genes related to the synthesis of fatty acids and succinate compounds in peony. Subsequently, they performed structural modeling and docking analysis on the selected genes. PoUGT75C1 was highly consistent with the template UGT structure (PDB code: 5u6s), with a sequence similarity of 39.28%, a GMQE value of 0.90, and a RMSD of 0.14 Å for the three-dimensional structural superposition, and had the same α-helix and β-chain regions. Docking results showed that 11 amino acids of PoUGT75C1 (GLN134, SER273, MET274, SER325, GLN327, TRP345, ASN346, SER347, GLU350, ASP366, and TRP324) may interact with UDP-Glc. TRP324, SER325, GLN327, and GLU350 were predicted to interact with uridine groups, while MET274, SER273, GLU350, and ASN346 were thought to interact with diphosphate groups. GLN134 and ASP366 interacted with glucose. The docking fraction of PoUGT75C1 with UDP-Glc was -8.26 kcal / mol. Substrate docking analysis showed that PoUGT75C1 interacts with He through 9 amino acid residues (TYR13, TYR79, TYR180, GLN276, ARG299, MET274, PRO14, THR15, and PHE70); interacts with Km through 5 residues (TYR13, TYR79, THR15, PHE70, and PHE43); and interacts with Lu through 8 residues (TYR13, TYR79, T...). The PoUGT75C1-UDP-Glc complex interacts with five substrates. Residues interacting with He (TYR180, GLN276, ARG299, PRO14, MET274, THR15, and PHE70) are listed. Nine residues interact with Qu (TYR13, TYR180, ARG299, SER301, PRO14, SER273, MET274, THR15, PHE70, and PHE43) and six residues interact with Sy (TYR13, TYR79, MET274, THR15, PHE70, and PHE43). Dosing scores of the PoUGT75C1-UDP-Glc complex with these five substrates showed the highest affinity for He (-8.057 kcal / mol), followed by Lu (-8.201 kcal / mol), similar to Km (-7.643 kcal / mol) and Qu (-7.656 kcal / mol), and the lowest affinity for Sy (-7.508 kcal / mol).

[0028] In summary, this invention ultimately screened a UGT gene, 169276_c0_g1, which is most closely related to AtUGT75C1 of the L subfamily of the Arabidopsis UGT superfamily, and named it the PoUGT75C1 gene. The function of this gene will be further verified and explained in the following embodiments.

[0029] The full-length nucleotide sequence of the PoUGT75C1 gene is shown in SEQ ID NO.1:

[0030] The nucleotide sequence of the CDS region of the PoUGT75C1 gene is shown in SEQ ID NO.2:

[0031] The amino acid sequence of the protein encoded by the PoUGT75C1 gene is shown in SEQ ID NO.3: MENRHHFLLVTYPVQSHINPALQFAKRLLRIGVHITYAISISARRRMTRGAPTPEGLDFVEFSDGYDDGFKPSGDVDRFMSELSRHGSQTLTDIIVSSANKGRPITCLVYTGLRLPWA AEVARVLHVPSALLWIQPATVLDIYYYYYNNEDYKNTIIDNSPLIELPGLPLLTKSDLPSFLDPSNDAHTFALPTFREQLEALAKETNPKILVNTFDALEPLALIAIEKLNLIAIGPL IPSAFLDGKDPSDTCFGGDLFQCAKGKNYIEWLNTKPVSSVIYLSFGSISILSKQQMEEIAQGLLQSGRPFLWVIRAKDQNGAEEKDLQDKLINCLEELEQHGLIVPWCSQVEVLSHP SLGCFVTTHCGWNSTTESLVCGVPVVAFPLWSDQGTNAKLVQDVWKTGVRVRAAKVGGIVEAGEINRCVEIIMGGGEKGEEMRRNAKKWKDLASEAVKEGGSSDKNLKAFVDGVVQGCY .

[0032] The sequence of the specific fragment used for gene silencing is shown in SEQ ID NO.4: .

[0033] The primers used in the following examples are shown in Table 1.

[0034] Table 1. Specific information about the primers Example 1 I. Experimental Methods 1. Plant materials Fengdan ( P. ostii Seeds were collected in 2021 from the Peony Resource Nursery of the Institute of Botany, Chinese Academy of Sciences, from the large-flowered yellow peony (Paeonia suffruticosa). P. ludlowii Seeds were collected in Lhasa, Tibet in 2025; wild-type Arabidopsis thaliana ( Arabidopsis thaliana, Columbia ) is preserved at the Institute of Botany, Chinese Academy of Sciences; Nicotiana benthamiana is preserved at the Institute of Botany, Chinese Academy of Sciences.

[0035] 2. RNA extraction RNA was extracted using a universal plant total RNA extraction kit (RNAprep Pure Plant Kit) under RNase-free conditions, following the kit instructions. After extraction, the concentration and purity were determined using an Implen ultra-micro spectrophotometer. The RNA was diluted to 250 ng / μL and stored at -80°C for later use.

[0036] 3. RNA reverse transcription Reverse transcription was performed using an All-in-one RT Master reverse transcription kit. The reaction system is shown in Table 2.

[0037] Table 2 RNA reverse transcription system The above reaction system was reacted in a PCR instrument at 50℃ for 5 min and 80℃ for 5 sec. The cDNA obtained after the reaction was stored in a -20℃ freezer for later use.

[0038] 4. Gene cloning The PoUGT75C1 gene was cloned using a high-fidelity PCR kit (1.1×S4 Fidelity PCR Mix) with cDNA from *Pteris vittata* seeds as a template. Primers used for gene cloning are shown in Table 1. The 50 μL reaction volume is shown in Table 3.

[0039] Table 3 Candidate UFGT gene cloning reaction system The following PCR amplification conditions were used: Pre-denaturation: 98℃, 2 min. Amplification: 98℃, 10 s; 57℃, 10 s; 72℃, 45 s; 35 cycles in total. Final extension: 72℃, 5 min. PCR products were detected by 1% agarose gel electrophoresis. Bands of the correct length were extracted using a DNA gel extraction kit.

[0040] Then, ligation and transformation were performed: 4 μL of the recovered product and 1 μL of pEASY-Blunt Simple Cloning Vector were added to a 0.2 mL tube, and the PCR was run at 37°C for 15 min. Add 5 μL of the product to 30-50 μL of T1 competent cells, gently tumble to mix, incubate on ice for 20-30 min, incubate at 42℃ for 30 s, immediately place on ice for 2 min, add 500 μL of LB medium to the tube, incubate at 200 rpm and 37℃ for 1 h, centrifuge at 1500 g for 1 min, discard part of the supernatant, mix the bacterial culture, spread evenly on solid medium containing antibiotics until the bacterial culture dries, seal, and incubate overnight at 37℃ in an inverted culture dish. Add 500 μL of LB medium containing Kans to a 1.5 mL centrifuge tube, pick white single colonies and add them to the centrifuge tube, shake at 200 rpm and 37℃ for at least 5 h until the bacterial culture is clear. After the bacterial culture becomes turbid, perform bacterial PCR using Taq enzyme according to the following reaction system: 2×DNA Taq Mix 5 μL, M13F (10 μM) 0.5 μL, M13R (10 μM) 0.5 μL. 1.0 μL of bacterial culture and 3 μL of ddH2O were used. The following PCR amplification conditions were applied: Pre-denaturation: 94℃, 3 min. Amplification: 94℃, 30 s; 55℃, 30 s; 72℃, 1 min; 30 cycles in total. Final extension: 72℃, 10 min. PCR products were detected by 1% agarose gel electrophoresis. Bacterial cultures with correct bands were sent to BioMed Sequencing for sequencing.

[0041] 5. Carrier Construction Overexpression vector construction: The pSUPER1300 vector with a GFP tag was used as the overexpression vector. The overexpression vector was double-digested with restriction endonucleases XbaⅠ and SacⅠ. The reaction system is shown in Table 4.

[0042] Table 4. Double enzyme digestion reaction system The reaction was carried out at 37℃ for 15 min, and the double enzyme digestion products were identified by gel electrophoresis and then recovered by gel extraction.

[0043] Using the complete CDS sequence of the PoUGT75C1 gene (SEQ ID NO.2) as a template, the homologous arm sequence was cloned using a high-fidelity enzyme. The target gene with homologous arms was ligated to the vector using the Uniclone One Step Seamless Cloning Kit. The primer sequences for constructing the homologous arms are shown in Table 1.

[0044] Thaw DH5α competent cells on ice. Add 50 μL of competent cells and 5 μL of ligation product to a centrifuge tube, gently swirl to mix, and immediately place on ice for 30 min. Heat shock the centrifuge tube in a 42°C water bath for 90 s, then quickly transfer it to an ice bath to cool the cells for 2 min, without shaking the centrifuge tube. In a clean bench, add 500 μL of LB liquid medium (antibiotic-free) to the centrifuge tube, mix well, and incubate at 37°C with shaking at 200 rpm for 1 h to revive the cells. In a clean bench, evenly spread the bacterial suspension onto LB solid medium containing Kan (100 μg / mL) until the suspension is completely absorbed. Inverted plates were incubated overnight at 37°C for 12-16 h. Single colonies were picked and transferred to centrifuge tubes containing 600 μL LB liquid medium (Kan, 100 μg / mL). After shaking and incubating for 5 h at 37°C and 200 rpm, bacterial PCR was performed. Successfully identified bacterial cultures were sent to Biomed Sequencing for sequencing.

[0045] Construction of prokaryotic expression vector: Prokaryotic expression was performed using the pGEX-4T-2 vector. The overexpression vector was double-digested with restriction endonucleases BamHI and SacI, using the same double digestion system as above. The ligation with the CDS sequence of the target gene and the transformation of DH5α competent cells were performed using the same method as above. The primers for cloning the homologous arm of the target gene are shown in Table 1.

[0046] Construction of virus-induced gene silencing (VIGS) vector: The target gene was transiently silenced using the TRV2 vector. The overexpression vector was double-digested with restriction endonucleases BamHI and EcoRI, using the same double digestion system as above. A specific fragment of the target gene, approximately 300 bp in length, was amplified and ligated into TRV2. Primers for cloning the specific fragment and homologous arm primers are shown in Table 1.

[0047] 6. Protein expression (1) Transform the protein-induced strain Rosetta with the plasmid containing the pGEX vector containing the target gene, and identify positive strains by PCR; (2) Take 2 mL of overnight culture and add it to a 500 mL Erlenmeyer flask containing 150 mL of LB culture medium (containing 50 μg / mL Amp). Incubate at 37°C and 200 rpm for about 2 hours until the OD value is reached. 600 = 0.5-0.8; (3) To explore the optimal induction conditions, two final IPTG concentrations were set: 0.2 mM and 0.4 mM; the induction temperatures were set at 16℃ and 28℃, the rotation speed was 120 rpm, and the induction time was 24 h. (4) Centrifuge at 4℃, 8000 rpm for 5 min to collect bacteria; (5) Add 15 mL of PBS (containing 1 mM PMSF benzoyl fluoride protease inhibitor: 100 μL of 100 mM stock solution added to 10 mL of PBS; 1 mM DTT: 10 μL of 1 M DTT stock solution added to 10 mL of PBS), and gently aspirate until the cells are evenly suspended. (6) On ice, use ultrasonic breaking for 15 min, power 10-20%, ultrasonic for 3 s, stop for 5 s, add 1 mM PMSF and DTT; (7) Centrifuge at 4℃ and 8000 rpm for 20 min; (8) Transfer the supernatant to a new centrifuge tube and add 1 mM PMSF and DTT.

[0048] 7. Protein purification Protein purification using column chromatography: (1) Prepare buffer: Equilibration / wash buffer: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4; Elution buffer: Prepare 10 mM reduced glutathione with equilibration buffer (prepare fresh), and add 10 mM DTT to the equilibration buffer and elution buffer. Filter the buffer with a 0.22 μm or 0.45 μm filter membrane before use; (2) Equilibration of glutathione-agarose medium: Gently invert the container containing GST-Bind packing material (GLUTATHIONESEPHAROSE 4B) to homogenize the resin. Prepare 1 mL of resin for every 100 mL of cell lysate, pack it into the chromatography column, and open the cap at the bottom of the purification column to allow the liquid inside the column to flow out naturally; (3) Add 5 times the volume of resin equilibration buffer to resuspend the equilibration medium, and repeat the equilibration process three times in total; (4) Add the supernatant obtained after the lysis step in “6. Protein Expression”, resuspend it evenly with the resin, and gently shake it on a side-shaking shaker at 4℃ for 30 min to allow it to fully combine. (5) Open the cap at the bottom of the purification column to allow the liquid in the chromatography column to flow out naturally. Collect and save the eluent for SDS-PAGE electrophoresis. (6) Add 10 times the volume of resin washing buffer, shake gently at 4°C for 15 min to wash away the impurities that are not bound to the resin. Repeat the washing process 4-5 times, collect and store the washing buffer for SDS-PAGE electrophoresis. (7) Add 0.5 mL of elution buffer to each mL of resin, incubate at room temperature for 15 min, collect and store the elution buffer, and repeat the elution 3-5 times. This elution buffer is the purified GST-labeled protein sample.

[0049] (8) Concentrate protein samples using an Amicon® Ultra filter (Merck millipore).

[0050] (9) Determine the protein content and perform SDS-polyacrylamide electrophoresis to detect the protein.

[0051] 8. In vitro enzyme activity test The enzyme reaction system (50 μL) contained 3 μg of purified protein, 2 mM UDP-Glc, 100 mM Tris-HCl, and 0.1 mM of each receptor substrate.

[0052] Determination of optimal pH for the reaction: Tris-HCl enzyme activity buffer solutions with pH values ​​of 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 were prepared. Using quercetin as the substrate, the reaction solutions were prepared according to the above reaction system, and the reactions were carried out at 37°C for 30 min. Each reaction was repeated 3-5 times.

[0053] Determination of the optimal reaction temperature: Using quercetin as the substrate, the reaction solution was prepared according to the above reaction system using Tris-HCl enzyme activity buffer at pH 7.0. Reactions were carried out at 25℃, 30℃, 37℃, 42℃, 50℃, 60℃, and 70℃ for 60 min each. Each reaction was repeated 3-5 times. After the above reactions were completed, 50 μL of methanol was added to terminate the reaction, and the results were analyzed using UPLC-DAD. The UPLC-DAD method was based on Dr. Yin Qinggang's dissertation (Identification and Functional Analysis of Flavonoid Glycosyltransferases in the Leguminosae Model Plants *Lotus Root* and Soybean*, 2016).

[0054] Enzyme kinetic parameters were determined under the optimal enzyme-catalyzed reaction conditions defined above. 3 μg of purified and concentrated recombinant protein was added to 25, 50, 75, 100, 125, 150, 200, and 400 μM flavonoid substrates, and the reaction was carried out for 60 min. Each protein was reacted in 3-5 replicates at each concentration. After the reaction was complete, 50 μL of methanol was added to terminate the reaction, and the results were analyzed using UPLC-DAD.

[0055] Enzyme kinetic parameters were calculated using Hyper 32 software, with the double reciprocal curve method (Lineweaver-Burkplots) selected for calculation. K m Value and V max Value, according to the formula k cat = V max Calculate the kcat value based on the amount of protein, then calculate... kcat / K m value.

[0056] 9. Transformation of Arabidopsis thaliana Arabidopsis thaliana Columbia ecotype (Col-0) was used as wild-type material and grown under culture conditions of 23℃, 16 h light / 8 h dark photoperiod, and 60% relative humidity. The Super1300::UFGT-GFP overexpression vector constructed in "5. Vector Construction" was transformed into Agrobacterium tumefaciens strain GV3101. This Agrobacterium tumefaciens strain GV3101 was then used to transform Col-0 inflorescences, and stable T3 generation transgenic lines were obtained through screening for subsequent analysis. The expression level of the target gene was detected by RT-qPCR, and fatty acid content was determined according to the method described in Dr. Li Linkun's dissertation ("Study on the PoKO gene for dwarfing oilseed lines in 'Fengdan' gibberellin synthesis, 2022"). The primer sequences used for RT-qPCR are shown in Table 1.

[0057] 10. Subcellular localization of tobacco Nicotiana benthamiana plants were cultured in pots at 23℃, with a 16-h light / 8-h dark photoperiod and approximately 60% relative humidity. The constructed overexpression vector Super1300::UFGT-GFP was combined with either the nuclear marker Super1300::NF-YA4-mCherry or the endoplasmic reticulum marker pCAMBIA1300::HDEL-mCherry, and co-infected with *Agrobacterium tumefaciens* strain GV3101. Three days after infection, the fluorescence signals of GFP and mCherry were detected using a confocal microscope (Olympus FV1000MPE, Japan).

[0058] 11. VIGS The TRV2-gene vector constructed in "5. Vector Construction" was transformed into Agrobacterium tumefaciens GV3101 and cultured. Infection treatment was performed approximately 40 days after pollination of *Peony peony* (seed filling stage). The specific process is as follows: (1) Micro-shaking of bacterial culture: Agrobacterium containing TRV1, TRV2 empty vector and TRV2- gene vector was cultured on YEB solid medium for three days. Single colonies were picked and shaken at 28℃ and 200rpm overnight. The target gene band was detected by PCR.

[0059] (2) Large-scale shaking of bacterial culture: Dilute the bacterial culture prepared the previous day at a ratio of 1:50 into fresh LB medium, shake 150 mL in a 500 mL Erlenmeyer flask, and incubate overnight at 28°C and 200 rpm. The amount of TRV1 culture shaken = the amount of TRV2 culture shaken = the sum of the amount of TRV-gene culture shaken.

[0060] (3) Preparation of infection solution: Add 1 mL of 1M MES, 400 μL of AS stock solution (concentration of 1%), and 1 mL of 1M MgCl2 to every 100 mL of distilled water, and adjust the pH to 5.6 with dilute hydrochloric acid.

[0061] (4) Collection of bacteria: Centrifuge at 5000 rpm for 10 min to collect bacteria, discard the supernatant, resuspend the bacteria in the infection solution, and mix thoroughly with a pipette until there are no lumps. Measure the concentration of the resuspended bacterial solution using a spectrophotometer, and adjust the OD value accordingly. 600 =1.0-1.5, mix TRV1:TRV2-gene vector = 1:1, TRV1:TRV2 = 1:1, and let stand in the dark for 3-6 hours.

[0062] (5) Seed treatment steps: Freshly collected seeds are soaked in the infiltration solution and the seeds are infiltrated by vacuum pumping. The vacuum is drawn to -15 psi and the infiltration is carried out for 40 min. After infiltration, the seeds are placed on moist filter paper and placed in the dark at 8℃ for 3 days, and then cultured in the culture room for 7-10 days.

[0063] (6) The expression level of the target gene was analyzed by RT-qPCR using SYBR qPCR Master Mix (Vazyme, China). The primer sequences used for VIGS and RT-qPCR are listed in Table 1.

[0064] (7) Harvest the seeds. After the mature seeds are naturally dried, separate the kernel and seed coat. Grind the seed coat into powder using a vibratory mill. Accurately weigh about 300 mg of seed coat powder and place it in a 2 mL centrifuge tube. Add 1.5 mL of 70% methanol aqueous solution (methanol volume: water volume = 70:30) to the centrifuge tube. Extract the solution using an ultrasonic device at 4℃ for 20 min, centrifuge at 12000 rpm for 12 min, and transfer the supernatant to a 2 mL centrifuge tube. Repeat three times. Filter the collected supernatant through a 0.22 μm filter membrane and determine the content of ursine compounds in the seed coat using a SHIMADZU LC-2070 / 2080 high performance liquid chromatograph. The chromatographic conditions are as follows: Column: Inersil ODS-SF C18 (4.6 mm × 250 mm, 1.8 µm); Mobile phase: Phase A, 0.1% formic acid aqueous solution; Phase B, acetonitrile; Injection volume: 10 µL; flow rate: 1.0 mL·min -1 The column temperature was set to 40°C. The diode array detector was scanned at 380 nm and 360 nm.

[0065] Gradient elution program: 0-10 min, 20% B; 10-60 min, 23% B; 60-120 min, 27-30% linear gradient B; 120-130 min, 20% B.

[0066] Qualitative and quantitative analysis: Accurately weigh the standard, dissolve it in 70% methanol, and dilute to a concentration of 1 mg / mL. Use the standard to qualitatively analyze the metabolites. The content of metabolites was analyzed using a semi-quantitative method. Quantification was performed using a standard curve plotted with Suffruticosol B standard. Seven concentrations (0.015625, 0.03125, 0.0625, 0.125, 0.25, 0.5, and 1 mg / mL) of standard solutions were prepared. The peak areas of the standard solutions at each concentration were extracted to construct the corresponding standard curves.

[0067] (8) Harvest the seeds and test the fatty acid content in the seeds according to the method described above.

[0068] II. Experimental Results 1. Cloning of the target gene The agarose gel electrophoresis results showed that the band size was basically consistent with the theoretical length, indicating that the target fragment had been effectively amplified.

[0069] 2. Induction and purification of PoUGT75C1 protein This invention successfully cloned the PoUGT75C1 gene into the pGXT prokaryotic expression vector, transformed it into Rosetta(DE3), and optimized the induction conditions. Two IPTG concentration gradients were set: 0.2 mM and 0.4 mM. The induction temperatures were 16℃ and 28℃, respectively, and the rotation speed was kept constant at 120 rpm. SDS-PAGE analysis results showed ( Figure 1 The optimal induction conditions for the PoUGT75C1 gene were 0.4 mMIPTG and 16℃. Western blot results confirmed successful induction of PoUGT75C1 protein expression. Further purification using Ni-NTA affinity chromatography yielded the recombinant protein, and the results showed that the target protein band size was consistent with expectations. Figure 2 Furthermore, the purity is high, meeting the requirements for subsequent enzyme activity studies (Table 5).

[0070] Table 5. Purified concentrations of candidate UFGTs proteins 3. In vitro enzyme activity analysis of PoUGT75C1 protein Using UDP-Glc as the sugar donor and Km, Qu, Lu, He, and Sy as acceptor substrates, an in vitro enzymatic reaction was carried out at 37°C and pH=7. The formation of glycosylated products was confirmed by HPLC analysis and comparison with standards. The results showed that PoUGT75C1 can catalyze the glycosylation of flavonol Km and flavonoid Lu. Figure 3 PoUGT75C1 exhibits significant kinetic differentiation characteristics for Km and Lu (Table 6). It demonstrates higher catalytic efficiency for Lu. K cat / K m =403.21 mM -1 s -1 This is mainly due to its strong substrate affinity. K m =46.14 μM) and a high catalytic rate ( K cat =18.61 s -1 While Km exhibits a higher catalytic turnover rate ( K cat =29.21 s -1 However, due to its relatively weak affinity ( K m =83.35 μM, resulting in an overall catalytic efficiency of (350.49 mM). -1 s -1 The value of is lower than that of Lu. This difference may reflect the specificity of the active site of this protein for binding to different flavonoid compounds, and the structural features of Lu may be more conducive to its correct positioning and stable binding in the binding region.

[0071] Table 6 Enzymatic kinetic parameters of candidate UFGTs 4. In vivo functional verification of PoUGT75C1 4.1 Subcellular localization of PoUGT75C1 To clarify the subcellular localization of the PoUGT75C1 gene, its overexpression vector was co-expressed with the nuclear marker gene (Super 1300::NF-YA4-mCherry) and the endoplasmic reticulum marker gene (pCAMBIA1300::HDEL-mCherry) in the epidermal cells of Nicotiana benthamiana leaves. Laser confocal microscopy revealed that the GFP signal of PoUGT75C1 was mainly localized in the nucleus and cytoplasm. Figure 4 ).

[0072] 4.2 Instantaneous Silence Verification Function To investigate the in vivo function of the PoUGT75C1 gene in the secondary metabolism of peony seeds, this invention used VIGS technology to silence the gene and verified the silencing efficiency by qRT-PCR. Based on this, the changes in stilbene compounds in the silenced lines were systematically analyzed to reveal the regulatory role of this gene in the metabolic network.

[0073] (1) VIGS Silent Efficiency Verification qRT-PCR results showed that, compared with the TRV2 empty vector control, the expression level of the PoUGT75C1 gene in the corresponding silencing line was significantly decreased: the expression level of PoUGT75C1 decreased to 39.36% of the control. Figure 5 This confirms that the VIGS system successfully achieved specific silencing of the target gene, providing reliable genetic material for subsequent metabolite analysis.

[0074] (2) Effects of gene silencing on the accumulation of stilbene compounds Indigo compounds are a class of non-flavonoid phenolic compounds with a stilbene basic skeleton (C6-C2-C6). The two benzene rings are connected by an ethylene bridge. They are important defense substances for plants against pathogens and environmental stresses, and are also known as phytotoxicants. According to the complexity of their structure, they can be divided into: (1) Monomeric indigo compounds: such as resveratrol and pinosylvin; (2) Oligomeric indigo compounds: including dimers (such as trans-ε-viniferin and Gnetin H), trimers (such as Suffruticosol AC), and more complex polymers.

[0075] The results of the determination of stilbene-based substances showed that ( Figure 6 Silencing of the PoUGT75C1 gene promoted the synthesis of RE and SB (1.27-fold and 1.10-fold higher than the control, respectively), suggesting that it may be involved in inhibiting the synthesis of RE and SB or promoting their further modification.

[0076] (3) Effect of gene silencing on fatty acid content Fatty acid content analysis showed that ( Figure 7 Silencing of the PoUGT75C1 gene has a significant effect on the accumulation of major fatty acids in peony seeds, such as palmitic acid (PA), stearic acid (SA), oleic acid (OA), linoleic acid (LA), and α-linolenic acid (ALA): silencing of PoUGT75C1 caused a significant increase in the content of all fatty acids.

[0077] 4.3 Heterologous overexpression verification function To further explore the biological function of the PoUGT75C1 gene in plants, this invention constructed a PoUGT75C1 overexpression vector and transformed it into Arabidopsis thaliana Col-0 ecotype, obtaining multiple stable transgenic overexpression lines (OE). The expression level of the target gene in each line was verified by qRT-PCR, and the types and contents of flavonoids in transgenic Arabidopsis seedlings (21 days) and mature seeds, as well as the composition and accumulation levels of fatty acids in seeds, were systematically analyzed to reveal the potential regulatory functions of these genes in flavonoid and lipid metabolism.

[0078] (1) Detection of PoUGT75C1 gene expression level in overexpression lines The relative expression levels of the corresponding genes in each overexpression line were detected by qRT-PCR. The results showed that ( Figure 8 Compared with the wild type (WT), the expression level of PoUGT75C1 overexpression lines was 6.06-91.77 times that of WT.

[0079] (2) Effects of overexpression of PoUGT75C1 on fatty acid metabolism in Arabidopsis thaliana PoUGT75C1 overexpression had a significant effect on seed fatty acid composition. Figure 9 The contents of C16:0, C18:0, C18:1, C18:2, C18:3, C20:1, C20:2 and C22:1 in all OE strains were significantly higher than those in WT.

[0080] In summary, this invention, through gene silencing and overexpression techniques, verified the regulatory mechanism of the PoUGT75C1 gene on stilbene compounds and fatty acids in plants. Specifically, silencing the PoUGT75C1 gene increased the accumulation of stilbene compounds (RE and SB), and silencing the PoUGT75C1 gene increased the content of fatty acids (PA, SA, OA, LA, ALA). Overexpression of the PoUGT75C1 gene increased the content of fatty acids (C16:0, C18:0, C18:1, C18:2, C18:3, C20:1, C20:2, C22:1), thereby promoting the accumulation of fatty acids in seeds.

[0081] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A PoUGT75C1 gene, characterized in that, Its CDS region nucleotide sequence is shown in SEQ ID NO.

2.

2. A protein encoded by the PoUGT75C1 gene according to claim 1, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

3.

3. A recombinant vector, characterized in that, The recombinant vector is a recombinant vector that overexpresses the PoUGT75C1 gene of claim 1, or a recombinant vector that silences the PoUGT75C1 gene of claim 1.

4. A recombinant microorganism containing the recombinant vector of claim 3.

5. The use of the PoUGT75C1 gene of claim 1, the protein of claim 2, the recombinant vector of claim 3, or the recombinant microorganism of claim 4 in any of the following: (1) Regulate the fatty acid content in plant seeds; (2) Preparation of formulations to regulate the fatty acid content in plant seeds; (3) Cultivate plants with high fatty acid content; (4) Regulate the content of erucic acid compounds in plant seed coats; (5) Preparation of formulations to regulate the content of zirconia compounds in plant seed coats; (6) Cultivate plants with high content of succinate compounds.

6. The application according to claim 5, characterized in that, The method for regulating the fatty acid content in plant seeds includes the step of transferring the PoUGT75C1 gene into plants and stably overexpressing it to increase the fatty acid content of plant seeds; or, the step of silencing the PoUGT75C1 gene in plants to increase the fatty acid content of plant seeds. Alternatively, the method for regulating the content of erucic acid compounds in plant seed coats may include the step of silencing the PoUGT75C1 gene in plants to increase the content of erucic acid compounds in plant seed coats.

7. The application according to claim 6, characterized in that, The fatty acids include C16:0, C18:0, C18:1, C18:2, C18:3, C20:1, C20:2, and C22:1; The succinyl compounds include resveratrol and Suffruticosol B.

8. The application according to any one of claims 5-7, characterized in that, The plants mentioned include peony and Arabidopsis thaliana.

9. A method for increasing the fatty acid content in plant seeds, characterized in that, The method includes the step of transferring the PoUGT75C1 gene into a plant and stably overexpressing it to increase the fatty acid content of plant seeds; or the step of silencing the PoUGT75C1 gene in a plant to increase the fatty acid content of plant seeds. The nucleotide sequence of the CDS region of the PoUGT75C1 gene is shown in SEQ ID NO.

2.

10. A method for increasing the content of succinate compounds in plant seed coats, characterized in that, The steps include the PoUGT75C1 gene described in silent plants to increase the content of strychnine compounds in plant seed coats; The nucleotide sequence of the CDS region of the PoUGT75C1 gene is shown in SEQ ID NO.2.

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